Transferases (EC 2) catalyze transfer of a functional group from one substance to another. Glycosyltransferases, a superfamily of enzymes, are involved in synthesizing the carbohydrate portions of glycoproteins, glycolipids and glycosaminoglycans. Specific glycosyltransferases synthesize oligosaccharides by the sequential transfer of the monosaccharide moiety of an activated sugar donor to an acceptor molecule. Hence, a “glycosyltransferase” catalyzes the transfer of a sugar moiety from its nucleotide donor to an acceptor moiety of a polypeptide, lipid, glycoprotein or glycolipid. This process is also known as “glycosylation”. A carbohydrate portion which is structural part of e.g. a glycoprotein is also referred to as “glycan”. Glycans constitute the most prevalent of all known post-translational protein modifications. Glycans are involved in a wide array of biological recognition processes as diverse as adhesion, immune response, neural cell migration and axonal extension. As structural part of glycoproteins glycans also have a role in protein folding and the support of protein stability and biological activity.
In glycosyltransferase catalysis, the monosaccharide units glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), galacturonic acid (GalUA) and xylose are activated as uridine diphosphate (UDP)-α-D derivatives; arabinose is activated as a UDP-β-L derivative; mannose (Man) and fucose are activated as GDP-α-D and GDP-β-L derivatives, respectively; and sialic acid (=Neu5Ac; =SA) is activated as a CMP derivative of β-D-Neu5Ac.
Many different glycosyltransferases contribute to the synthesis of glycans. The structural diversity of carbohydrate portions of glycoproteins is particularly large and is determined by complex biosynthetic pathways. In eukaryotes the biosynthesis of the glycan-part of glycoproteins takes place in the lumen of the endoplasmatic reticulum (“ER”) and the Golgi apparatus. A single (branched or linear) carbohydrate chain of a glycoprotein is typically a N- or an O-linked glycan. During post-translational processing, carbohydrates are typically connected to the polypeptide via asparagine (“N-linked glycosylation”), or via serine or threonine (“O-linked glycosylation”). Synthesis of a glycan, no matter whether N- or O-linked (=“N—/O-linked”) is effected by the activity of several different membrane-anchored glycosyltransferases. A glycoprotein may comprise one or more glycan-connected amino acids (=“glycosylation sites”). A specific glycan structure may be linear or branched. Branching is a notable feature of carbohydrates which is in contrast to the linear nature typical for DNA, RNA, and polypeptides. Combined with the large heterogeneity of their basic building blocks, the monosaccharides, glycan structures exhibit high diversity. Furthermore, in members of a particular glycoprotein species the structure of a glycan attached to a particular glycosylation site may vary, thus resulting in microheterogeneity of the respective glycoprotein species, i.e. in a species sharing the same amino acid sequence of the poypeptide portion.
A sialyltransferase (=“ST”) is a glycosyltransferase that catalyzes transfer of a sialic acid (=5-N-acetylneuramic acid=Neu5Ac=NANA) residue from a donor compound to (i) a terminal monosaccharide acceptor group of a glycolipid or a ganglioside, or (ii) to a terminal monosaccharide acceptor group of an N—/O-linked glycan of a glycoprotein. For mammalian sialyltransferases including human ST species there is a common donor compound which is cytidine-5′-monophospho-N-acetylneuraminic acid (=CMP-Neu5Ac=CMP-NANA). Transfer of a sialic acid residue is also referred to as “sialylating” and “sialylation”.
In the glycan structure of a sialylated glycoprotein the (one or more) sialyl moiety (moieties) is (are) usually found in terminal position of the oligosaccharide. Owing to the terminal, i.e. exposed position, sialic acid can participate in many different biological recognition phenomena and serve in different kinds of biological interactions. In a glycoprotein more than one sialylation site may be present, i.e. a site capable of serving as a substrate for a sialyltransferase and being an acceptor group suitable for the transfer of a sialic acid residue. Such more than one site can in principle be the termini of a plurality of linear glycan portions anchored at different glycosylation sites of the glycoprotein. Additionally, a branched glycan may have a plurality of sites where sialylation can occur.
According to current knowledge, a terminal sialic acid residue can be found (i) α2→3 (α2,3) linked to galactosyl-R, (ii) α2→6 (α2,6) linked to galactosyl-R, (iii) α2→6 (α2,6) linked to N-acetylgalactosaminidyl-R, (iv) α2→6 (α2,6) linked to N-acetylglucosaminidyl-R, and (v) α2→8/9 (α2,8/9) linked to sialidyl-R, wherein —R denotes the rest of the acceptor substrate moiety. Hence, a sialyltransferase active in the biosynthesis of sialylconjugates (=“sialylation”) is generally named and classified according to its respective monosaccharide acceptor substrate and according to the 3, 6 or 8/9 position of the glycosidic bond it catalyzes. Accordingly, in the literature known to the art, e.g. in Patel R Y, et al, Glycobiology 16 (2006) 108-116, reference to eukaryotic sialyltransferases is made such as (i) ST3Gal, (ii) ST6Gal, (iii) ST6GalNAc, or (v) ST8Sia, depending on the hydroxyl position of the acceptor sugar residue to which the Neu5Ac residue is transferred while forming a glycosidic bond. Reference to sialyltransferases in a more generic way can also be made e.g. as ST3, ST6, ST8; thus, “ST6” specifically encompasses the sialyltransferases catalyzing an α2,6 sialylation.
The disaccharide moiety β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine (=Galβ1,4GlcNAc) is a frequent terminal residue of the antennae of N-linked glycans of glycoproteins, but may be also present in O-linked glycans and in glycolipids. The enzyme β-galactoside-α2,6-sialyltransferase (=“ST6Gal”) is able to catalyze α2,6-sialylation of a terminal Galβ1,4GlcNAc of a glycan or a branch of a glycan (=“antenna”). For general aspects thereof, reference is made to the document of DallOlio F. Glycoconjugate Journal 17 (2000) 669-676. In human and in other mammals there appear to be several species of ST6Gal. The present disclosure particularly deals with human β-galactoside-α-2,6-sialyltransferase I (=hST6Gal-I; EC 2.4.99.1 according to IUBMB Enzyme Nomenclature), but is not limited thereto.
The ST6 group of sialyltransferases comprises 2 subgroups, ST6Gal and ST6GalNAc. The activity of ST6Gal enzymes catalyzes transfer of a Neu5Ac residue to the C6 hydroxyl group of a free galactosyl residue being part of terminal Galβ1,4GlcNAc in a glycan or an antenna of a glycan, thereby forming in the glycan a terminal sialic acid residue α2→6 linked to the galactosyl residue of the Galβ1,4GlcNAc moiety. The resulting newly formed terminal moiety in the glycan is Neu5Acα2,6Galβ1,4GlcNAc.
The wild-type polypeptide of human β-galactoside-α-2,6-sialyltransferase I (hST6Gal-I) at the time of filing of the present document was disclosed as “UniProtKB/Swiss-Prot: P15907.1” in the publically accessible NCBI database (http://www.ncbi.nlm.nih.gov/protein/115445). Further information including coding sequences are provided as hyperlinks compiled within the database entry “Gene ID: 6480” (http://www.ncbi.nlm.nih.gov/gene/6480).
Mammalian sialyltransferases share with other mammalian Golgi-resident glycosyltransferases a so-called “type II architecture” with (i) a short cytoplasmic N-terminal tail, (ii) a transmembrane fragment followed by (iii) a stem region of variable length and (iv) a C-terminal catalytic domain facing the lumen of the Golgi apparatus (Donadio S. et al. in Biochimie 85 (2003) 311-321). Mammalian sialyltransferases appear to display significant sequence homology in their catalytic domain.
Donadio S. et al. expressed several N-terminally truncated variants of hST6Gal-I in CHO cells and found that N-terminal deletions comprising the first 35, 48, 60 and 89 amino acids yielded mutant enzymes which nevertheless were still active in transferring sialic acid to exogenous acceptors.
Glycosylation is an important posttranslational modification of proteins influencing protein folding, stability and regulation of the biological activity. The sialyl mojety (=sialic acid, 5-N-acetylneuramic acid, Neu5Ac) is usually exposed at the terminal position of N-glycosylation and therefore, a major contributor to biological recognition and ligand function, e.g. IgG featuring terminal sialic acids were shown to induce less inflammatory response and increased serum half-life.
The use of glycosyltransferases for enzymatic synthesis of defined glycan structures is becoming a tool to direct N-glycosylation of therapeutic proteins such as antibodies. Since glycosyltransferases of prokaryotic origin usually do not act on complex glycoprotein structures, sialyltransferases of mammalian origin are preferred. For example, Barb et al. (2009) prepared highly potent sialylated forms of the Fc fragment of immunoglobulin G using isolated human ST6Gal-I. However, the access to recombinant ST6Gal-I for therapeutic applications is still limited due to low expression and/or poor activity in various hosts (Pichia pastoris, Spodoptera frugiperda and E. coli).
It is known to the art that mammalian glycosyltransferases can be used advantageously for in vitro sialylating antennal acceptor sites of a complex target molecule such as a glycoprotein or a glycolipid. However, there still is a lack of means and methods to perform sialylation in a quantitatively controlled fashion, particularly with regards to target molecules with two or more acceptor sites capable of being sialylated. That is to say, it is desirable to provide means, methods and conditions allowing to sialylate just one out of several acceptor sites, as opposed to sialylating two or more, or even all acceptor sites of the target molecule. It is further desirable to control in a batch sialylation reaction mixture the balance between single-event sialylation of the target molecule and multiple event sialylation thereof.
Surprisingly, it was found initially that the two truncated variants Δ89 hST6Gal-I and Δ108 hST6Gal-I displayed different sialylation activities in-vitro on antibodies, but also on other target molecules. Apparently, the IgG-Fc antennal glycan G2 has two galactose moieties at the termini of the branches which can be sialylated. Under identical reaction conditions, Δ89 hST6Gal-I preferably catalyzes the synthesis of bi-sialylated glycans (G2+2SA), whereas the variant Δ108 hST6Gal-I synthesizes mono-sialylated glycans (G2+1SA). Therefore, both enzymes are suitable tools for the selective sialylation of glycan structures, particularly of N-glycans.
The original finding by the present inventors is that the Δ108 hST6Gal-I variant of human β-galactoside-α-2,6-sialyltransferase I is mainly capable of adding only a single sialyl residue to a target molecule with two or more antennal acceptor sites. In contrast, the enzymatic activity of human β-galactoside-α-2,6-sialyltransferase I and specific variants thereof such as Δ89 hST6Gal-I leads to multiple sialylation of the target molecule. Thus, by way of using Δ108 hST6Gal-I alone, for example in a batch process, a mono-sialylated target molecule can be produced from a target molecule with two or more non-sialylated antennal acceptor sites.
This paves the way for a number of different approaches, particularly in the field of in vitro glycoengineering of immunoglobulins, and also of other glycosylated target molecules. Here specifically and exemplarily a method is provided resulting in the production of mono-sialylated and bi-sialylated immunoglobulin G molecules, wherein the ration thereof is controlled by way of controlling the amounts of (i) Δ108 hST6Gal-I enzymatic activity and (ii) enzymatic activity of a human β-galactoside-α-2,6-sialyltransferase I capable of sialylating the acceptor sites of the target molecule in a saturating way. However, a number of other in vitro sialylation approaches with quantitative sialylation control of the target molecule to be sialylated become feasible and can be deduced from the present disclosure.
In a specific embodiment this document discloses the high-yield expression of two different variants of human β-galactoside-α-2,6-sialyltransferase I (hST6Gal-I, EC 2.4.99.1; data base entry P15907) by transient gene expression in HEK293 cells with yields up to 100 mg/L. The two variants were found to feature surprisingly distinct sialylation activities.